S4-S5 Linker Regulates RyR2 Channel Gating through Multiple Interactions

Abstract

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel in the sarcoplasmic reticulum of cardiac muscle and plays an important role in excitation-contraction coupling. Although recent cryo-EM studies provided near-atomic structures of type 1 ryanodine receptor (RyR1), molecular mechanism of the channel opening remains largely unknown. The S4-S5 linker (S45L) is an α-helical structure connecting the S4 and S5 transmembrane segments and mediates signal transmission in a wide variety of channels. To address the role of S45L in the RyR2 channel gating, we systematically mutated residues in S45L (Thr4751-Asn4762) and neighboring transmembrane segments (S5 and S6). The mutant RyR2 was stably expressed in HEK293 cells, and the channel activity was investigated by ER luminal Ca2+ measurements and [3H]ryanodine binding. Using high sequence identity between RyR1 and RyR2 (65% in the whole molecule and 93% in the core channel domain after S4), we constructed structural model of RyR2 based on the structures of RyR1, and the phenotypes of mutants were interpreted by the model. Plausible interactions between amino acid residues in S45L and neighboring domains that may regulate channel gating will be discussed.